Each of the references cited herein, including the references numbered 18, 25 and 57 to 200, is incorporated herein by reference.
1. Field of the Invention
The present invention relates generally to molecular biology and, more specifically, to methods of modulating L-selectin and P-selectin mediated binding in a subject by administering heparin to the subject in an amount that does not produce substantial anticoagulation activity or undesirable bleeding.
2. Background Information
L-selectin, E-selectin and P-selectin mediate the initial adhesive events directing the homing of lymphocytes into lymphoid organs, as well as the interactions of leukocytes and other inflammatory cells with endothelium at sites of inflammation. L-selectin is expressed on leukocytes, E-selectin is expressed on endothelium and P-selectin is expressed on platelets and endothelium. The three selectins bind to specific carbohydrate structures on opposing cells, for example, L-selectin binds to platelets and endothelium, whereas P-selectin and E-selectin bind to leukocytes.
Selectin adhesion is involved in disorders such as pathologic reperfusion injury, inflammatory disorders and autoimmune disorders. Selectin interactions also can mediate primary adhesive mechanisms involved in the metastasis of certain epithelial cancers. Thus, selectins are potential therapeutic targets for the treatment of pathologies characterized by undesirable or abnormal interactions mediated by selecting.
Much work has been directed to finding small carbohydrate molecules for use as competitive inhibitors to block selectin mediated interactions. For example, the tetrasaccharide sialyl-Lewisx (SLex) is recognized by all three selecting, and is a component of many naturally occurring high affinity selectin ligands, for example, the myeloid cell ligand for P-selectin, called PSGL-1. However, the interaction of selectins with purified SLex is weak and SLex demonstrates little selectivity among the selectins. Thus, while SLex and related structures may provide some therapeutic use, they are limited in being weak and nonselective inhibitors of selectin binding. Furthermore, they are very expensive to produce in the quantities required for treatment.
Heparan sulfates (HS) are naturally occurring glycosaminoglycan (GAG) chains that have diverse biological functions, generally mediated by their ability to interact with growth factors, receptors and the like. Heparin, which is a heavily modified GAG, is a heterogeneous mixture of long, unbranched carbohydrate chains consisting of repeating disaccharide units composed of uronic acids alternating with glucosamine residues which can be extensively modified.
Due to its anticoagulant activity, heparin is used clinically as an antithrombotic agent for treating human subjects having a disorder resulting from the abnormal or undesirable activation of the blood clotting cascade. Heparin""s anticoagulant activity is the result of a modified pentasaccharide sequence that is present on certain heparin chains and binds to antithrombin III, a regulatory protein of the clotting cascade. Heparin chains that are longer than about 18 saccharide units and that have the modified pentasaccharide sequence can enhance the ability of antithrombin III to bind to and inhibit the function of coagulation factors, thereby inhibiting the blood clotting cascade. Pharmaceutical preparations of heparin are enriched for antithrombin III binding chains, but also contain a mixture of other components.
L-selectin and P-selectin interact with a variety of sulfated compounds including heparan sulfates porcine intestinal mucosal (PIM) heparin and its fragments. Although L-selectin and P-selectin similarly bind to HS chains and PIM-heparin, they are different from E-selectin, which fails to bind the HS chains or PIM-heparin. The observation that L-selectin binding to endothelial cell HS chains is calcium dependent indicates that this interaction is similar to selectin""s interaction with natural ligands. This suggestion is supported by the ability of small heparin fragments to compete with L-selectin and P-selectin binding to SLex.
Pharmaceutical preparations of heparin are similar to crude commercial PIM-heparin. Thus, it might be expected that pharmaceutical heparin would have been used for inhibiting the binding of L-selectin and P-selectin to ligands present on cells in humans. However, heparin has not been used for the purpose of inhibiting L-selectin and P-selectin binding in humans because of concerns about potential undesirable side effects associated with its anticoagulant activity.
To address the problem of undesirable anticoagulant activity, low molecular weight (LMW) oligosaccharides, which are derived from heparin, but lack anticoagulant activity, have been prepared. When injected into mice, such LMW heparins can inhibit inflammation by binding to L-selectin and P-selectin. However, the cost of preparing LMW forms of heparin combined with the cost of new product testing to obtain FDA approval for use in humans is often prohibitive. In addition, it is unclear how efficacious the LMW heparins would be in inhibiting L-selectin and P-selectin binding when administered to a human subject. Thus, a need exists for developing methods of using readily available pharmaceutical compositions to inhibit L-selectin and P-selectin mediated binding in a subject without producing undesirable side effects. The present invention satisfies this need and provides related advantages as well.
The present invention provides a method of inhibiting L-selectin and P-selectin binding in a subject by administering to the subject an amount of heparin that does not produce substantial anticoagulant activity or undesirable bleeding. Thus, the invention provides methods of administering heparin in amounts that result in attaining a concentration less than about 0.2-0.4 units heparin per ml of plasma in the subject, such levels which inhibit L-selectin or P-selectin mediated binding in the subject. The invention further provides methods of treating a subject having an L-selectin or P-selectin related pathology by administering heparin in an amount that does not produce substantial anticoagulant activity or undesirable bleeding, for example, an amount that results in a concentration less than about 0.2-0.4 units heparin per ml of plasma in the subject.